Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons
© Vasefi et al.; licensee BioMed Central Ltd. 2013
Received: 17 January 2013
Accepted: 6 May 2013
Published: 14 May 2013
N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2–24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) β receptors which can protect neurons against NMDA-induced neurotoxicity.
In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered.
In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.
Keywords5-HT7 NMDA Hippocampus Isolated neurons Phosphorylation Trafficking
N-methyl-D-aspartate (NMDA) receptors are tetrameric channels composed of two NR1 and two NR2 or NR3 subunits . In the hippocampus, most NR2 subunits are either NR2A or NR2B  and there is evidence that heterotrimeric NMDA receptors containing both NR2A and NR2B are also present . Several studies have examined the ability of serotonin (5-HT) receptors to modulate NMDA receptor activity. For example, in isolated cortical neurons, activation of 5-HT1A receptors inhibits NMDA receptor currents  and 5-HT3 receptor activation reduces NMDA receptor currents in cortical slices . In contrast, in Xenopus oocytes, 5-HT2 receptor activation increases NMDA receptor currents  and in prefrontal cortical slices, 5-HT2A/2C agonists enhance NMDA-evoked responses .
Although first identified in the suprachiasmatic nucleus, 5-HT7 receptors are expressed throughout the CNS, including the hippocampus . The effect of 5-HT7 receptor ligands on NMDA-evoked currents remains unknown however recent studies provide clear evidence for the regulation of glutamatergic signaling by 5-HT7 receptors. 5-HT7 receptors inhibit NMDA-induced neurotransmitter release in the dorsal raphe nucleus (DRN) and the physiological role of 5-HT7 receptors in circadian rhythms is associated with an inhibition of glutamate-dependent events [9, 10]. In the suprachiasmatic nucleus, glutamate excitatory post-synaptic potentials (EPSPs) and glutamate-induced intracellular calcium levels are both inhibited by 5-HT7 receptor activation [11, 12]. Taken together, these studies suggest that 5-HT7 receptor activation decreases NMDA and/or glutamate receptor signaling. In contrast, compared to wild-type, 5-HT7 receptor knock-out mice display a reduced induction of long-term potentiation (LTP), magnitude of LTP, and hippocampus-associated learning . Therefore, although there is evidence that 5-HT7 receptors negatively regulate NMDA/glutamate signaling, deletion of 5-HT7 receptors decreased the magnitude of NMDA receptor-dependent events such as LTP.
5-HT7 receptors are Gαs–coupled, although they may couple to additional Gα isoforms including Gα12[14, 15]. Recently we identified the 5-HT7 receptor as a regulator of platelet-derived growth factor (PDGF) β receptor expression and activity . Activation of PDGFβ receptors by PDGF-BB selectively inhibits NR2B-containing NMDA receptor currents and this may be involved in the mechanism of PDGFβ receptor-mediated neuroprotection . Intriguingly, 5-HT7 receptor-induced upregulation of the PDGFβ receptor was sufficient to protect neurons against NMDA-induced excitotoxicity . Thus, we proposed that long-term activation of 5-HT7 receptors initiates pathways that ultimately negatively regulate NMDA receptor signaling.
To clarify the direct effects of 5-HT7 receptor activation on NMDA receptor signaling we examined the effects of 5-HT7 receptor agonists and antagonists on NMDA-evoked currents, NMDA receptor subunit phosphorylation, and subunit cell surface expression in the hippocampus. In isolated hippocampal neurons, application of the 5-HT7 receptor agonist, 5-CT, resulted in a rapid and sustained increase in peak NMDA-evoked currents. 5-HT7 receptor agonist treatment also differentially altered NMDA receptor subunit phosphorylation and cell surface expression. These data, along with our previous work, suggest a model for differential NMDA receptor regulation by 5-HT7 receptors over the short- and long-term.
5-CT treatment differentially alters NMDA receptor subunit phosphorylation
NMDAR subunit/phosphorylation site
Treatment (5 min)
5-CT + SB
NR1/896, n = 8
2.07 ± 0.39**
0.91 ± 0.31
1.06 ± 0.28
NR1/897, n = 8
1.51 ± 0.13**
1.00 ± 0.39
0.95 ± 0.11
NR2A/total, n = 10
0.71 ± 0.07**
0.97 ± 0.37
0.86 ± 0.17
NR2B/1472, n = 5
2.79 ± 0.37**
1.49 ± 0.61
2.86 ± 1.33
NR2B/1252, n = 10
1.39 ± 0.49
1.50 ± 0.23
1.53 ± 0.31
NR2B/1336, n = 7
1.25 ± 0.19
1.98 ± 0.31
2.36 ± 0.36**
5-HT7 receptors are involved in aspects of learning and memory associated with hippocampal function . For example, 5-HT7 receptor knock-out mice display impaired contextual fear-conditioning  and display a reduced ability to recognize new environments . These and other studies have promoted interest in 5-HT7 receptors as a potential drug target in Alzheimer’s disease and are further supported by a study demonstrating an increase in memory formation by a 5-HT7 receptor agonist, AS 19 . NMDA receptors are crucial components of learning and memory pathways in the hippocampus. Thus, the positive linkage between 5-HT7 receptors and NMDA receptor activity may explain how 5-HT7 receptors promote learning and memory as well as their involvement in LTP.
Since all three 5-HT7 receptor splice variants identified in rats are positively coupled with adenylate cyclase and display some level of constitutive activity , it is not surprising that the activation of 5-HT7 receptors in hippocampal slices increases the phosphorylation of the NR1 subunit at serine 897. On the NR2B subunit, tyrosine 1472 is required/involved in CaMKII binding and activation  and this phosphorylation site is linked to spinal pain transmission . Interestingly, 5-HT7 receptors agonists promote pain after formalin injection in animal models  however others have found anti-nociceptive effects of 5-HT7 receptors [37, 38]. These differences may be due to distinct central and peripheral pain pathways that both contain 5-HT7 receptors.
In summary, we have now identified two pathways downstream of the 5-HT7 receptor that ultimately regulate NMDA receptor activity/signaling (Figure 6). We have demonstrated that the upregulation of PDGFβ receptors by long-term treatment with 5-HT7 receptor agonists is sufficient to protect hippocampal neurons against NMDA excitotoxicity [16, 18] whereas acute activation of 5-HT7 receptors increased NMDA-evoked currents. These findings may help to explain why previous reports identified 5-HT7 receptors as both positive and negative regulators of NMDA receptor signaling.
Materials and methods
Reagents and antibodies
5-CT, LP 12 (4-(2-Diphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1- piperazinehexanamide hydrochloride), and H89 were purchased from Sigma (St. Louis, MO, USA). The 5-HT7 receptor antagonists SB 258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzene sulfonamide) and SB 269970 (R-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol as well as Go 6983 were purchased from Tocris (Ellisville, MO, USA). Antibodies purchased from Millipore (Bellerica, MA) include anti-NR1, anti-phospho-896-NR1, anti-phospho-897-NR1, anti-NR2A, anti-phospho-pan-NR2A, anti-NR2B, anti-phospho-1252-NR2B, anti-phospho-1336-NR2B, and anti-phospho-1472-NR2B.
Cell isolation and whole-cell recording
CA1 neurons were isolated from hippocampal slices of postnatal day 14–21 Wistar rats as previously described . The extracellular solution was composed of 140 mM NaCl, 1.3 mM CaCl2, 25 mM N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (HEPES), 33 mM glucose, 5.4 mM KCl, and 0.5 μM tetrodotoxin, and 0.5 μM glycine, with pH of 7.3-7.4 and osmolarity ranging from 320–330 mOsm. Recordings were done at room temperature. The intracellular solution consisted of 11 mM ethyleneglycol-bis-(α-amino-ethyl ether) N,N’-tetra-acetic acid (EGTA) as intracellular calcium chelating buffer, 10 mM HEPES, 2 mM MgCl2, 2 mM tetraethyl ammonium chloride (TEA-Cl) to block K + channel, 1 mM CaCl2, 140 mM CsF, and 4 mM K2ATP. NMDA currents were evoked by rapid application of NMDA solution delivered from a multi-barreled fast perfusion system for 2 s every minute.
Hippocampal slices were prepared, treated with drugs for 5 min, and homogenized chilled lysis buffer (20 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 30 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 0.5% SDS, and 1% triton-X-100; supplemented with Halt Protease and Phosphatase Inhibitor (Thermo, Fisher, Markham, Ontario)) prior to use. Lysates were centrifuged at 14,000 × g for 20 min at 4°C and the supernatant was collected. The supernatant was subjected to SDS-PAGE and proteins were transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk in Tris-buffered saline and 0.1% Tween for 1 h at room temperature or overnight at 4°C, and incubated in primary antibodies for 1 h at room temperature or overnight at 4°C. Membranes were washed three times in Tris-buffered saline with 0.1% Tween-20, incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, washed again, and bound antibodies were visualized by the enhanced chemiluminescence using the chemiluminescent substrate (Millipore, Etobicoke, Ontario). Images of Western blots were taken on the Kodak 4000 MM Pro Imaging Station, and densitometric analyses were performed using the Kodak Molecular Imaging software. Membranes were then stripped and reprobed with other antibodies.
Surface biotinylation assay
Hippocampal slices were incubated for 5 min with 5-HT7 receptor agonists and antagonists. Slices were washed in ice-cold ECF and incubated with 0.5 mg/ml Sulfo-NHS-LC-biotin (Pierce) for 30 min. The biotin reaction was quenched by washing with 10 mM glycine. Slices were washed twice more and homogenized in lysis buffer. Lysate protein concentrations were normalized and lysates were incubated with streptavidin beads overnight at 4°C (Sigma). Beads were washed three times in lysis buffer and boiled in loading buffer for 5 min before separation by SDS-PAGE.
Statistical analysis of the data was performed using the Prism® GraphPad program. For electrophysiology, graphs and sample tracings were made using the Origin® program. Significance level was set at α = 0.05. Data was analyzed by one-way ANOVA or Student’s t-test where appropriate.
All animal experiments were performed in agreement with the guidelines of the policies on the Use of Animals at the University of Waterloo (protocol #09-17) and the University of Western Ontario.
Ddorsal Raphe nucleus
Excitatory post-synaptic potentials
Platelet-derived growth factor
Special thanks to Nancy Gibson and Dawn McCutcheon for their animal care and use of their facility while ours was under construction. This research was funded through generous start-up funding from the University of Waterloo, Faculty of Science and by the National Science and Engineering Research Council of Canada. The contributions of KY and JFM to this manuscript were supported by the Canadian Institutes of Health Research.
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